Our research is based on the hypothesis that effective non-invasive imaging of tumor hypoxia will improve cancer care, and that it is important to better understand the mechanistic basis of tumor hypoxia images. Thus, we propose to develop and evaluate different non-invasive hypoxia imaging methods in laboratory and clinical studies. In Research Project 1 (RP1) we shall examine the biological underpinning of hypoxia imaging using a xenograft model that contains a hypoxia-inducible reporter gene, the expression of which can be visualized by optical and PET imaging. With it, the initial hypoxia-induced molecular event and its spatial distribution can be visualized and compared to other tumor hypoxia surrogates, e.g. expression of downstream genes and the trapping of exogenous hypoxia markers. Our goal is to use this model as a tool to validate the use of three hypoxic cell markers: 18F-FMISO, 1241-IAZG and 18F-EF5. In RP2 we shall assess the efficacy of several NMR approaches: T19F-FMISO, DCE-MRI and lactate measurement, in comparison with microPET imaging of exogenous hypoxia markers and pO2 probe data. In addition, both NMR and microPET methods will be evaluated for studies on the dynamics of tumor hypoxia, either intrinsic or resulting from intervention, e.g. radiation or anti-angiogenesis treatment. RP3 will validate a radio-gene therapy strategy to overcome hypoxia-mediated radioresistance, combining hypoxia imaging, delivery of vectors with a radiosensitizing effecter gene, verification of delivery by molecular imaging, and tumor eradication by radiation. We shall design/construct vectors that express a hypoxia-inducible reporter and an effecter gene that antagonizes DMA damage repair, and generate replication-defective and conditionally-replicative adenovirus. The efficacy of adenovirus delivery and the radiosensitizing effect of these genes will be studied in vitro and in vivo. Finally, we shall localize hypoxic volumes in xenografts, deliver the adenovirus under image guidance, and evaluate the efficacy of this strategy. RP4 is a clinical PET/CT study, testing 1241-IAZG and 18F-FMISO in colorectal and head/neck cancers. In the first study series we shall perform head-to-head comparison of the two tracers in -100 patients per disease site to evaluate both prognostic value and image quality, in order to identify the best tracer for each disease site. In the second study series, hypoxia images of patients with early colorectal cancers will be compared with pO2 probe measurement and immunohistochemistry of endogenous markers. We believe that this translation research program, integrating laboratory and clinical investigation on an important question in cancer management, will eventually lead to improvement in cancer treatment outcome.